Heparin and Heparan Sulfate
Heparan sulfate is a member of the glycosaminoglycan family of polysaccharides. These glycosaminoglycans are linear polymeric carbohydrate chains that are the glycan part of proteoglycan molecules that are important structural and functional components of the extracellular matrix.
Heparan sulfate and heparin like compounds share a biosynthetic pathway, and are characterized by a repeating disaccharide units consisting of a 2-amino-2-deoxy-D-glucose
Heparan sulfate and heparin like compounds share a biosynthetic pathway, and are characterized by a repeating disaccharide units consisting of a 2-amino-2-deoxy-D-glucose (glucosamine, GlcN) residue linked .alpha.-(1-4) to a uronic acid residue in a repeating fashion. The glucosamine can exist in the 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-deoxy-2-sulfamino-D-glucose (GlCNS) and 2-deoxy-2-sulfamino, 6-O-sulfate-D-glucose (GlcNS6S) forms. The uronic acid residue can be either .beta.-D-glucuronic acid (GlcA) or .alpha.-iduronic acid (IdoA), the latter of which can exist in the 2-O-sulfated form (IdoA2S). The exact composition of heparan sulfates varies significantly depending on the source and even the specific stage of cell growth. Heparin has been shown to be a more extensively transformed biosynthetic form of heparan sulfate. Heparin possesses a greater proportion of the IdoA2S and GlcNS6S residues and is therefore a more highly sulfated substance. In-vivo heparin is found in association with mast cells, and not with the extracellular matrix of vascularized tissue, as is heparan sulfate.
Heparin is a commonly employed anticoagulant and antithrombotic drug. Commercial heparin is usually isolated from porcine or bovine mucosa, or bovine lung tissue. The anticoagulant action of heparin has been shown to reside largely in its ability to interact with and potentiate the activity of a circulating protein, antithrombin III (AT-III). AT-III is a serine protease inhibitor that inhibits many of the serine proteases involved in the coagulation cascade, particularly thrombin (Factor IIa) and Factor Xa. Heparin interacts with AT-III to form a complex that inhibits thrombin and Xa much more effectively than AT-III alone. Heparin interacts with AT-III through a specific high affinity pentasaccharide sequence that represents only a minor portion of any heparin chain. Heparin also interacts with another serine protease inhibitor, heparin cofactor II, to potentiate the inhibition of thrombin. The interaction with HC-II operates through different structural requirements and does not appear to have the same degree of specificity as the AT-III interaction.
The potent anticoagulant related activities of heparin, unfortunately, preclude its use for many of the other potential indications. In particular, the anticoagulant activity of heparin can lead to hemorrhage and bleeding complications when administered at doses that yield other therapeutic benefit. This factor has led to an interest in selectively decoupling the anticoagulant activity of heparin from other biological properties of the molecule. This decoupling strategy can also be applied to selectively decoupling or retaining other desirable biological and therapeutic properties. More selective agents would have reduced risk of toxic and/or contra-indicative effects. Also, the specificity could enhance overall bioavailability and potency of the agent relative to heparin.
Heparin/heparan sulfate have been shown to interact with a large and expanding number of proteins, and have been associated with numerous biological functions and activities. This poly-pharmacy is mediated by the ability of heparin-like materials to bind and inhibit or stimulate the action of a myriad of extracellular matrix resident and circulating receptors, proteins and enzymes. The broad pharmacological action of heparin and related compounds are of considerable therapeutic interest for a large number of indications, particularly in cardiovascular related diseases. There is considerable precedent for the utility of heparin based therapeutics for disease states related to hemostasis and thrombosis, and a recently expanding interest in conditions related to atherosclerosis, angiogenesis, metastasis and inflammation.
There is a fairly large effort in developing heparin based therapies for treating thrombotic and proliferative vascular disorders. However, despite considerable in-vitro, and anecdotal in-vivo evidence there has been relatively less focus on the anti-inflammatory use of heparin derived materials. Some of the anti-inflammatory properties of heparin derived compounds include; inhibition of complement activation, inhibition of heparanase dependent T-lymphocyte migration, inhibition of leukocyte platelet interaction. In addition, heparin and derivatives have shown activity in animal models of DTH, EAE, ischemia reperfusion and asthma, as well as anecdotal description of benefit in humans suffering from asthma and ischemia (myocardial infarction).
As mentioned above, its potent anticoagulant activity makes unattractive its therapeutic use for cardiovascular disorders. In particular, the anticoagulant activity of heparin leads to hemorrhage and bleeding complications when administered in therapeutic doses.
Pathology of shock
In general, shock can be described as widespread hypoperfusion of cells and tissue due to reduction in blood volume or cardiac output or redistribution of blood resulting in an inadequate effective circulating volume. Shock is usually classified into four types: (1) Cardiogenic; (2) Hypovolemic; (3) Septic and (4) Neurogenic, largely on the basis of the hemodynamic derangement that leads to the condition. Extensive study has resulted in an understanding of the general mechanisms that lead to the pathology associated with shock. However, the myriad of biochemical processes that mediate the pathology are only just beginning to be understood. This relates to a complex cascade of inflammation-related events that have been studied in association with hypovolemic and septic shock that result in toxic injury to cell membranes of endothelial and other organ cells, leukocytes and platelets, activation of intrinsic and extrinsic coagulation pathways, complement activation and formation of the vasoactive and chemotactic fragments C5a and C3a.
Hypovolemic shock, (2), and models for studying this condition are described by Chaudry and Ayala in "Immunological Aspects of Hemorrhage" (R. G. Landes Co., Austin, Tex., 1992). Generally, hypovolemic shock due to reduced blood flow associated with blood loss results in "sludging" of the blood and capillary "plugging" by erythrocytes, platelets and neutrophils. This in turn leads to the insufficient delivery of oxygen and nutrients to cells and tissues, deficient clearance of other metabolites, and to activation of neutrophils and platelets. This oxidant stress (hypoxia) and release of other factors from the endothelium and macrophages stimulates the arachidonic acid cascade and the production of chemoattractant and inflammation mediators, leading to further neutrophil infiltration. The activation of neutrophils, platelets, macrophages and the complement cascade leads to the release of numerous biologically active agents including cytokines. These factors stimulate expression of adhesion molecules on the surface of the endothellum, neutrophils and leukocytes which permit binding and ultimately migration of the neutrophils and leukocytes through the extracellular matrix (ECM) and basement membrane of blood vessels and capillaries. This migration or extravasation is attributed to the action of a number of extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases and endoglycosidases (i.e. heparanases), which are released by activated neutrophils, leukocytes and/or platelets. The damage to the ECM and basement membrane results in increased vascular permeability, and infiltration of organs by neutrophils and leukocytes.
An analogous series of events is associated with septic shock except, and most critically, the key mediators of the inflammatory response are unlikely to be the same as those that cause hypovolemic shock. The initial blood volume reduction in septic shock occurs as a result of blood pooling after endotoxin stimulate neutrophil activation and the release of inflammation mediating cytokines (INF, IL-1 and IL-6, IL-10, TGF-.beta., etc.).
It is important to keep in mind that hypovolemic and septic shock are distinct diseases. Hypovolemic shock is a general collapse of the circulatory system that can be caused by many events including any trauma to the circulatory system (e.g. gun shot wound, automobile injury, burns, stabbing, and so on). Septic shock, on the other hand, is caused by bacterial infection. Thus, as mentioned above, the causes of these diseases are highly likely to be distinct.
Ischemia/reperfusion injury (I/RI) is another instance where inflammation mediated cell and organ damage result after a reduced blood flow state (ischemia).
The vascular damage associated with hypovolemic shock, and the resulting infiltration of neutrophils and leukocytes into the various organs leads to tissue damage and ultimately multiple organ failure (MOF) and acute respiratory distress syndrome (ARDS). The destructive agents and mediators are numerous and include cytokines, enzymes and various other inflammatory agents. MOF and ARDS can occur in severe shock and often result in death. For therapeutic agents to be effective in shock, they must protect the microvasculature and various organs (liver, kidney, heart, spleen and gut) from failure. The importance of protecting or restoring gut function and intestinal function in hemorrhagic shock and I/R injury has been reported, and correlates with reduced septic complications, and long-term survival.